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. 2011 Apr;39(8):3128-40.
doi: 10.1093/nar/gkq1251. Epub 2011 Jan 11.

An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA

Fan Zhang  1 , Alan G Hinnebusch
Affiliations

An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA

Fan Zhang et al. Nucleic Acids Res. 2011 Apr.

Abstract

Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisiae has revealed the translation of upstream open reading frames that initiate with near-cognate start codons in many transcripts. Two such non-translation initiation codon (AUG)-initiated upstream open reading frames (uORFs) (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1-4) that regulate GCN4 translation. We verified that nAuORF2 is translated in vivo by demonstrating β-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its non-AUG initiation codon (AUA) start codon with the non-cognate triplet AAA, whereas translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in non-starved cells, nor on its derepression in response to histidine limitation, nutritional shift-down or treatment with rapamycin, hydrogen peroxide or methyl methanesulfonate. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, or dependent on Gcn2, the sole eIF2α kinase of yeast, in histidine-deprived cells. Thus, although nAuORF2 is translated in vivo, it appears that this event is not stimulated by eIF2α phosphorylation nor significantly influences GCN4 translational induction under various starvation or stress conditions.

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Figures

Figure 1.
Figure 1.
Locations of nAuORFs 1 and 2 and the four conventional uORFs in the GCN4 mRNA leader. The sequence is shown numbered relative to the ATG (+1) of the Gcn4 coding sequence. Open reading frames are enclosed with rectangles, and both the ATG, or near-cognate start codons, and the stop codon are boxed within each rectangle and the start codons are also underlined. The nucleotide substitution and insertion mutations introduced to remove the nAuORF stop codon and introduce a BamHI site for constructing the nAuORF-lacZ fusion are indicated above the sequence, and the naturally occurring BstEII site exploited for cloning purposes is also indicated.
Figure 2.
Figure 2.
Evidence that nAuORF2 is translated in vivo from its AUA near-cognate start codon. (A) Schematic of GCN4-lacZ and nAuORF-lacZ constructs. (1) The nAuORFs 1 and 2 and uORFs 1–4 are depicted schematically in the leader of the wild-type GCN4-lacZ allele (on plasmid p180) drawn approximately to scale, indicating the TTG and ATA triplets that encode the UUG and AUA start codons of nAuORFs 1 and 2, respectively. (2–3) Variants of GCN4-lacZ containing point mutations in the AUG codons of the four uORFs (construct 2, on plasmid p227) and also in the non-AUG codons of the two nAuORFs (construct 3, on plasmid pLfz482), shown as ‘X’s that eliminate the cognate uORFs/nAuORFs. (4) The uORF1-lacZ construct (on plasmid p466). (5–8) Wild-type and mutant versions of the nAuORF-lacZ constructs (on plasmids pLfz489, pLfz491, pLfz493 and pLfz495, respectively) depicted as described above. (B and C) nAuORF-lacZ expression requires the AUA start codon of nAuORF2. Transformants of GCN4 strain H2833 harboring the indicated lacZ constructs on single-copy plasmids described in panel A were cultured in SC-Ura to saturation, diluted into fresh SC-Ura and grown for 6 h to an A600 of ∼1 (U, for uninduced), or for 2 h in SC-Ura-His and then an additional 6 h with 10 mM 3-AT (I, for induced). WCEs were prepared and assayed for β-galactosidase, measured in units of nmol of ONPG cleaved min−1 mg−1 of protein. The results obtained from three independent transformants were averaged and the mean and SEM values plotted.
Figure 3.
Figure 3.
Eliminating the near-cognate start codons of nAuORFs 1 and 2 has no effect on regulation of Gcn4 protein levels in non-starved cells, histidine-limited cells, or during nutritional shift-down. (A) Schematics of GCN4 alleles. The wild-type (1) or mutant (2–8) GCN4 alleles under examination, depicted as described in Figure 2A (contained on plasmids p164, p237, p238, pLfz450, pLfz453, pLfz456, pLfz469 and pLfz470, respectively). (B and C) Substituting the start codons of nAuORF1 and 2 has no effect on complementation of gcn4Δ by mutant GCN4 alleles. Transformants of gcn4Δ strain H2835 harboring the indicated GCN4 alleles described in (A) were cultured in SC-Ura to saturation and serial 10-fold dilutions were spotted on SC-Ura plates or SC-Ura,-His plates supplemented with 15 mM or 30 mM 3-AT (and excess leucine to exacerbate the Gcn− phenotype) and incubated at 30°C for 2–3 days. Essentially identical results were obtained for an independent set of transformants for these groups of constructs (data not shown). (D and E) Substituting the start codons of nAuORF1 and 2 has no effect on regulated expression of Gcn4 protein in response to histidine starvation or nutritional shift-down. (D) Histidine starvation. Strains described in (B–C) were cultured as described in Figure 2 for assaying β-galactosidase, except that they were induced with 3-AT for only 2 h. WCEs were prepared under denaturing conditions by extraction with tricholoracetic acid and aliquots representing equal proportions of total WCE (or 2X of this amount) were resolved by SDS–PAGE and subjected to western analysis using antibodies against Gcn4 or, to provide a loading control, the eIF2B subunit Gcd6, which is not under GCN4 control. Triangles depict loading of 1X and 2X amounts of the same WCE in successive lanes. (E) Nutritional shift-down. Strains described in (B–C) were cultured in SC-Ura to A600 of 0.8–1.0 and aliquots were collected by centrifugation, resuspended in SD and incubated for 20 min prior to harvesting. WCEs were prepared and subjected to western analysis as in (D). U, uninduced; I, induced by 3-AT; N, non-starved; S, starved by nutritional shift-down.
Figure 4.
Figure 4.
Eliminating the near-cognate start codons of nAuORFs 1 and 2 has no effect on regulated GCN4-lacZ expression in non-starved or histidine-limited cells. (A) Schematics of GCN4-lacZ alleles. The wild-type (1) or mutant (2–7) GCN4-lacZ alleles under examination, depicted as described in Figure 2A (contained on plasmids p180, p226, pLfz460, pLfz463, pLfz466, pLfz473 and pLfz474, respectively). (B and C) Regulation of GCN4-lacZ expression is not altered by mutations in the nAuORF start codons. Transformants of GCN4 strain H2833 harboring the indicated lacZ constructs described in (A) were cultured in the absence (U) or presence (I) of 3-AT and WCEs were assayed for units of β-galactosidase, as described in Figure 2B and C.
Figure 5.
Figure 5.
Mutating the near-cognate start codons of nAuORFs 1 and 2 to non-cognate triplicates has no effect on GCN4-lacZ expression under various stress conditions. (A) Schematics of the GCN4-lacZ alleles under examination, depicted as in Figure 2A, contained on plasmids p180 and pLfz474, respectively. (B) Transformants of yeast strain H2833 harboring GCN4-lacZ constructs described in (A), were cultured in SC-Ura (Un-treated) or in SC-Ura and treated with 2 mM hydrogen peroxide (H2O2) for 2 h, 200 ng/ml rapamycin for 4 h or 0.07% (v/v) methyl methanesulfonate (MMS) for 1 h. WCEs were assayed β-galactosidase activity as described in Figure 2B and C.
Figure 6.
Figure 6.
Histidine limitation imposed with 3-AT does not substantially induce initiation at near-cognate start codons. (A) nAuORF-lacZ fusion. Transformants of GCN2 strain H2833 or isogenic gcn2Δ strain H2931 harboring the wild-type nAuORF-lacZ construct on plasmid pLfz489 were cultured and analyzed for β-galactosidase expression as described in Figure 2B and C. (B) HIS4-lacZ fusion. Transformants of GCN2 strain H2833 harboring a plasmid-borne HIS4-lacZ fusion containing an ATG start codon (p367) or TTG start codon (p391) were cultured and analyzed for β-galactosidase expression as described in Figure 2B and C, and the ratio of enzyme activities observed for the TTG to ATG fusions was determined and plotted.
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